In a separator electrode assembly microbial fuel cell, oxygen crossover from the cathode inhibits current generation by exoelectrogenic bacteria, resulting in poor reactor startup and performance. To determine the best approach for improving startup performance, the effect of acclimation to a low set potential (-0.2V, versus standard hydrogen electrode) was compared to startup at a higher potential (+0.2V) or no set potential, and inoculation with wastewater or pre-acclimated cultures. Anodes acclimated to -0.2V produced the highest power of 1330±60mWm-2 for these different anode conditions, but unacclimated wastewater inocula produced inconsistent results despite the use of this set potential. By inoculating reactors with transferred cell suspensions, however, startup time was reduced and high power was consistently produced. These results show that pre-acclimation at -0.2V consistently improves power production compared to use of a more positive potential or the lack of a set potential. © 2013 Elsevier Ltd.